SEMA6A drives GnRH neuron-dependent puberty onset by tuning median eminence vascular permeability

Innervation of the hypothalamic median eminence by Gonadotropin-Releasing Hormone (GnRH) neurons is vital to ensure puberty onset and successful reproduction. However, the molecular and cellular mechanisms underlying median eminence development and pubertal timing are incompletely understood. Here we show that Semaphorin-6A is strongly expressed by median eminence-resident oligodendrocytes positioned adjacent to GnRH neuron projections and fenestrated capillaries, and that Semaphorin-6A is required for GnRH neuron innervation and puberty onset. In vitro and in vivo experiments reveal an unexpected function for Semaphorin-6A, via its receptor Plexin-A2, in the control of median eminence vascular permeability to maintain neuroendocrine homeostasis. To support the significance of these findings in humans, we identify patients with delayed puberty carrying a novel pathogenic variant of SEMA6A. In all, our data reveal a role for Semaphorin-6A in regulating GnRH neuron patterning by tuning the median eminence vascular barrier and thereby controlling puberty onset.

Defective GnRH neuron migration and/or innervation of the ME leads to reduced GnRH secretion, and consequently delayed or absent puberty.These are the hallmarks of reproductive disorders such as Congenital Hypogonadotropic Hypogonadism (HH), and also contribute to the etiology of familial Delayed Puberty (DP) 7,8 .The genetic determinants underlying these reproductive disorders remain unknown in many congenital cases, suggesting the existence of mechanisms controlling the development and function of the GnRH neuronal system relevant to these conditions 9,10 .
Semaphorin-6A (SEMA6A) is a transmembrane semaphorin mainly known for its roles during axon guidance 25,26 , neuronal positioning 27,28 and angiogenesis 29 .To exert its biological functions, SEMA6A signals as a ligand through Plexin-A2 and Plexin-A4 receptors 28,30,31 .Consistent with a role in the GnRH neuronal system, SEMA6A is expressed in the developing mouse olfactory system and hypothalamus 32,33 .However, its function in the GnRH system or in mechanisms underlying puberty onset are unknown.
Here, by combining mouse and human genetics with tailored in silico, in vitro and in vivo models, we unveil a role for SEMA6A in GnRH neuron innervation and ME vascular permeability, and identify SEMA6A as an additional player in the regulation of puberty timing.

SEMA6A is expressed along GnRH neuron migration route during brain development
To investigate the potential role of SEMA6A in GnRH neuron development and function, we first studied SEMA6A expression in wild-type (WT) mouse tissues at stages relevant for GnRH neuron development and function.Double immunofluorescence staining in the nasal compartment at embryonic day (E) 12.5 and E14.5, corresponding to the start and peak of GnRH neuron migration, respectively, showed colocalization of SEMA6A and Peripherin, a TN marker (Fig. 1a, b).In contrast, GnRH neurons in the nasal compartment, identified by anti-GnRH staining, did not express SEMA6A at E12.5 and E14.5 (Fig. 1c, d).Similarly, no SEMA6A expression was found in OECs, marked with an anti-Brain lipid-binding protein (BLBP) antibody (Fig. 1e, f).At E14.5, when the majority of GnRH neurons are scattered in the MPOA of the hypothalamus along TN fibers 14,34 , SEMA6A was strongly expressed in this region but it did not co-localize with GnRH + neurons or Peripherin + TN fibers (Supplementary Fig. 1a, b).Next, expression of SEMA6A was examined at E18.5, when GnRH neurons have attained their final position in the hypothalamus and start to project to the ME 35 .Although GnRH + cell bodies in the MPOA and GnRH + neurites in the ME did not express SEMA6A (Fig. 1g, h), strong and punctate SEMA6A expression was detected in the ME in close proximity to GnRH + axonal terminals (Fig. 1h).Our observations in mouse embryos were confirmed in human embryonic tissue at Carnegie Stage (CS) 19, corresponding to E12.5 in mouse, showing SEMA6A co-localization with axons in the nasal parenchyma (Fig. 1i-m).
Together, these data show that during GnRH neuron migration from the nasal compartment to the MPOA SEMA6A initially localizes in TN fibers, while during late gestation SEMA6A is expressed in close proximity to GnRH + axons in the ME.

Loss of SEMA6A does not affect GnRH neuron migration but reduces GnRH innervation of the ME
Our expression analysis suggests that SEMA6A may regulate GnRH neuron migration along nasal axons either directly or indirectly, by controlling nasal axon development, consistent with its well-characterized role as an axon guidance cue 25,26 .To explore this model, nasal axon patterning and GnRH neuron migration were analyzed in E14.5 Sema6a +/+ and Sema6a -/-mouse embryos using immunohistochemistry, as described previously 14 .Unexpectedly, no obvious defects in the patterning of Peripherin + axons were observed in Sema6a -/-embryos as compared to Sema6a +/+ littermates (Fig. 2a).Also, no significant differences in the number and positioning of GnRH neurons were found (Fig. 2b, c and Supplementary Table 1).
Since the GnRH system appeared intact at E14.5, we next analyzed (1) the final positioning of GnRH neurons in the MPOA, and (2) ME innervation in E18.5 embryos, when GnRH axons can be found at or near the ME 35,36 .Although Sema6a -/-and Sema6a +/+ mice displayed a similar number of GnRH neurons in the forebrain (FB) (Fig. 2d and Supplementary Table 1), GnRH neuron innervation of the ME was significantly reduced in Sema6a -/-mice by 2-fold (Fig. 2e, f; p = 0.0189; two-tailed unpaired Student's t test).However, the canonical SEMA6A receptors Plexin-A2 and Plexin-A4 28,30,31 (Supplementary Fig. 2a) were not detected in MPOA-resident GnRH neurons by immunolabelling (Supplementary Fig. 2b, c).This suggests that SEMA6A does not modulate ME innervation by acting on GnRH neurons but rather through functions in other cell types.
Together these findings indicate that SEMA6A is dispensable for GnRH neuron migration and positioning, but is required for innervation of the ME by GnRH + axons.
Female and male mice lacking SEMA6A exhibit delayed puberty onset and sexual maturation defects Proper innervation of the ME by GnRH axon terminals during development is crucial to ensure GnRH secretion and thereby puberty onset and reproductive function at postnatal stages 1,35 .To analyze whether SEMA6A expression in the ME is maintained postnatally, we assessed SEMA6A localization relative to GnRH axon terminals in the ME of pre-(P24) and post-(P60) pubertal mice.Interestingly, strong punctate SEMA6A expression was observed in close proximity to GnRH axon terminals in the ME at both stages (Fig. 3a, b).This suggests that SEMA6A might also play a role in maintaining the GnRH system.
Since SEMA6A was strongly expressed in the ME just before puberty (P24), we next investigated the role of SEMA6A in pubertal timing.The day of vaginal opening (VO), a proxy measurement for mouse pubertal activation of the HPG axis 35,37,38 , was assessed in Sema6a +/+ , Sema6a +/-and Sema6a -/-female mice.Sema6a -/-females displayed significantly delayed VO compared to WT mice, with 3 out of 9 Sema6a -/-females displaying a closed vaginal phenotype at the moment of termination at P45, 54 and 57 (red dots in Fig. 4a and Supplementary Fig. 3a).VO of Sema6a -/-mice was delayed by 12.1 ± 2.6 days (mean difference ± SEM) compared to Sema6a +/+ mice, and by 5.9 ± 0.9 days when the 3 female mice still displaying a closed vagina at the time of sacrifice were excluded (Fig. 4a; Sema6a +/- p = 0.9376, Sema6a -/-p < 0.0001 and delayed Sema6a -/-only p < 0.0001 vs. Sema6a +/+ ; One-way ANOVA followed by Dunnett's post-hoc test).Sema6a -/-mice had significantly increased weight at VO as they were on average older than their WT littermates at puberty onset (Supplementary Fig. 3b).In subsequent studies, we assessed the appearance of the first estrous (FE) after VO, which corresponds to the first ovulation and indicates the reaching of sexual competence 35 .We confirmed delayed VO in this new group of mice (Fig. 4b; Sema6a +/- p = 0.5186 and Sema6a -/-p = 0.0002 vs. Sema6a +/+ ; Two-way ANOVA followed by Dunnett's post-hoc test) and detected a significant delay in FE 12.9 ± 2.1 days compared to Sema6a +/+ ; Fig. 4b and Supplementary Fig. 3c; Sema6a +/-p = 0.5550 and Sema6a -/-p < 0.0001 vs. Sema6a +/+ ; Two-way ANOVA followed by Dunnett's post-hoc test.One female out of 9 had not reached FE at the time of termination (P58, 24 days after VO) (Fig. 4b, red triangle).
To assess whether the reduced ME innervation observed at E18.5 persisted at later stages, we next analyzed GnRH axon terminals in the ME of adult Sema6a -/-mice.Consistent with the observed delayed puberty phenotype, all Sema6a -/-females displayed a 2.6-fold decrease in ME innervation compared to age-matched Sema6a +/+ females (Fig. 4d, e; p = 0.0311; two-tailed unpaired Student's t test).Notably, two Sema6a -/-female mice that failed to achieve VO (Fig. 4a) displayed a stronger reduction in ME innervation as compared to Sema6a -/-mice exhibiting delayed VO.Similar to our observations at E18.5, the number of GnRH neurons in the MPOA was unaffected in adult Sema6a -/- females (Supplementary Table 1; p = 0.3258).Similarly, Sema6a -/-males had a significant 2.2-fold decrease in ME innervation compared to agematched Sema6a +/+ mice (Fig. 4d, f; p = 0.0389; two-tailed unpaired Student's t test).
We further found that adult Sema6a -/-females exhibited smaller ovaries but normal folliculogenesis (Fig. 4g-i; ovary weight: p = 0.0049; follicle subtype number/total number: primary p = 0.7666, secondary p = 0.6837, pre-antral p = 0.3500, early antral p = 0.8205; two-tailed unpaired Student's t).Sema6a -/-males displayed decreased gonadal size and a reduced number of Leydig cells, as detected by Cytochrome P450 17A1 (CYP17A1) immunostaining (Fig. 4j-l; testis weight: p = 0.0182; CYP17A1 + area: p = 0.0002; two-tailed unpaired Student's t).Consistent with this observation, Lhb mRNA levels were significantly reduced in Sema6a -/-pituitary glands (Fig. 4m; p = 0.0161).Finally, to assess general fertility, breeding records of Sema6a mutant mice were compared to those of WT C57Bl/6J mice for a period of 9 months (Supplementary Table 2).In this period, Sema6a +/-males bred with Sema6a +/-females produced fewer litters compared to the breeding of WT mice (68.6% successful in Sema6a +/-vs.80.3% successful in WT).Crosses of Sema6a -/-males with either Sema6a +/-or WT females were only successful in 35% of the cases.Out of 9 Sema6a -/- males in our colony, 4 were able to produce a litter, which occurred in only 50% of their matings.Overall litter size was not affected in the different crosses.These data support the other changes found in the reproductive system of Sema6a mutants described above.
Together, these results show a requirement for SEMA6A in the innervation of the ME by GnRH neurons, and in the regulation of puberty onset and sexual maturation.

SEMA6A is expressed by oligodendrocytes in the proximity of blood capillary loops
To establish the mechanisms through which SEMA6A controls GnRH neuron innervation and puberty onset, we first defined the cellular source of SEMA6A at the ME.Interestingly, SEMA6A signal was prominent in the basal part of the ME, which is rich in fenestrated blood capillary loops 39 .These mediate the delivery of GnRH to the pituitary, but also the entry of cues from peripheral tissues that fine tune ME homeostasis and consequently modulate GnRH structural plasticity [40][41][42] .Hence, we evaluated SEMA6A localization relative to blood vessels by immunolabelling SEMA6A in combination with endothelial cell (EC) 12 marker Isolectin B4 (IB4).Interestingly, SEMA6A was not detected in ECs but was concentrated and aligned to ME fenestrated blood capillary loops (Fig. 5a).To identify the source of SEMA6A in the ME, additional staining with markers for different cell types (secretory neurons, astrocytes, β-tanycytes and oligodendrocytes (OLs)) involved in ME homeostasis were performed 43,44 .SEMA6A was not detected in the Synaptophysin + terminals of neurosecretory neurons (Fig. 5b), but surrounded these structures, as shown for GnRH-enriched terminals.Further, SEMA6A did not localize in Glial fibrillary acidic protein (GFAP) + astrocytes (Fig. 5c) or in Vimentin + β-tanycytes (Fig. 5d).Instead, and in agreement with previous reports showing that SEMA6A is required for OL differentiation 45 , SEMA6A was detected in Oligodendrocyte transcription factor 2 (Oligo2) + cells (Fig. 5e).SEMA6A selectively localized to Glutathione S-transferase P1 (Gst P1) + maturing OLs but not oligodendrocyte precursor cells (OPCs) that expressed Platelet-derived growth factor receptor alpha (PDGFRα) (Supplementary Fig. 4a, b).
These findings therefore suggest a possible role for SEMA6A produced by maturing OLs in the regulation of ME vascular permeability, which is crucial for proper GnRH homeostasis 5,6,39 .

Abluminal SEMA6A induces vascular permeability
To explore the hypothesis that SEMA6A regulates ME vascular permeability, we tested the ability of SEMA6A to modulate the barrier function of EC monolayers.Trans-endothelial electrical resistance (TEER) was measured in response to SEMA6A stimulation from either the apical (luminal) or basal (parenchymal) side 47 (Fig. 6a).Although SEMA6A is produced as a transmembrane protein, its extracellular domain can be released to generate a secreted ectodomain 48 .Therefore, we transfected COS-7 cells with a vector encoding mouse SEMA6A ectodomain (SEMA6A ECTO ) and used conditioned medium to stimulate ECs.
The ability of SEMA6A ECTO to affect permeability was initially examined in human umbilical vein endothelial cells (HUVECs), a wellestablished human model of ECs, whose sensitivity to hyperpermeability-inducing agents was confirmed by the significant TEER reduction induced by apical exposure to vascular endothelial growth factor A (VEGFA), a well-known potent inducer of vascular hyperpermeability [49][50][51] (Supplementary Fig. 5a).Exposure of the apical surface of the HUVEC monolayer to SEMA6A ECTO did not affect TEER (Supplementary Fig. 5b; SEMA6A ECTO apical vs mock apical, 60 min p = 0.0011, 120 min p = 0.9998, 180 min p = 0.9810; Two-way ANOVA followed by Tukey's post-hoc test).In contrast, TEER was significantly reduced, and hence permeability was increased, following treatment of the basal compartment with SEMA6A ECTO , as compared to HUVECs treated with control medium (Supplementary Fig. 5b; SEMA6A ECTO basal vs mock basal: 60 min p = 0.0365, 120 min p = 0.0006, 180 min p < 0.0001; Two-way ANOVA followed by Tukey's post-hoc test).This indicates that SEMA6A can selectively induce vascular permeability in an EC monolayer.
This indicates that parenchymal, but not luminal, SEMA6A acts as a potent inducer of endothelial permeability in vitro.

SEMA6A regulates ME capillary fenestration and vascular permeability in vivo
Having found that parenchymal SEMA6A was able to bind PLXNA2 on the abluminal side of EC monolayers in vitro to promote endothelial permeability suggests that in vivo SEMA6A, which is expressed in close proximity of blood capillary loops (Fig. 5a), may play a role in regulating the ME vascular barrier.In agreement with this hypothesis, PECAM1 + capillary loops in the ME expressed Plexin-A2 (Fig. 7a).Further, transcardial perfusion of Evans Blue (EB) dye, to mimic peripheral signals diffusing through the ME barrier in vivo, led to EB extravasation in the ME of control mice.In contrast, EB leakage was significantly less in Sema6a -/-MEs (Fig. 7b, c; p = 0.0212; two-tailed unpaired Student's t test).To assess whether this reduction in ME barrier permeability was associated with structural changes, the number of Plasmalemma Vesicle Associated Protein (PLVAP) + fenestrated capillary loops was quantified [56][57][58] .Notably, Sema6a -/-mice had significantly fewer PLVAP + loops invading the ME parenchyma, as compared to WT mice (Fig. 7d, e; p = 0.023; two-tailed unpaired Student's t test).Double staining for PLVAP and PECAM1 showed reduced PLVAP immunostaining and a decrease in PECAM1 + capillary loops in the ME of Sema6a -/-mice (Supplementary Fig. 6a).This indicates that reduced ME barrier permeability caused by SEMA6A loss is accompanied by defective structural remodeling of ME capillaries.The morphology of Vimentin + β-tanycytes, known to modulate NGE unit plasticity and consequently GnRH secretion 17 , was unchanged in Sema6a -/-mice (Supplementary Fig. 6b).
Together, these findings show that SEMA6A is required in vivo to maintain ME barrier permeability and the formation of fenestrated capillary loops in the ME.

Exome sequencing of patients with DP identifies a loss-offunction SEMA6A variant in six individuals
In view of the observation that Sema6a -/-mice present with delayed pubertal timing, we interrogated exome sequencing data from a large cohort of patients with self-limited DP and identified a potentially pathogenic, variant in SEMA6A (ENSG00000092421, NCBI gene ID: 57556).This variant (NM_020796; c.1268 T > C; p.I423T) was carried by six affected individuals from our cohort, including four members of one family, and all individuals were heterozygous for this variant (Fig. 8a).The presence of this SEMA6A variant in probands and family members was confirmed by Sanger sequencing (Supplementary Fig. 7).The affected subjects did not carry any other predicted pathogenic variants in known HH/DP causing genes nor other pituitary hormone deficiencies were excluded.
All the individuals (both males and females) carrying the SEMA6A variant showed classical clinical and biochemical features of self-limited DP, with delayed onset of Tanner stage 2 and delayed peak height velocity.Proband A is a male who presented at 14.5-15.0years with testicular volumes of 3.7 mL, bone age delayed by 3.6 years and short stature (height SD score −2.75) (see Table 1 for detailed auxological data).He had normal olfaction and no evidence of chronic illness.His short stature was related to pubertal delay, as height SDS at both 6.5-7.0 years and at adult height was −1.3, in keeping with his midparental target height.His delayed puberty was inherited from his mother, who had menarche aged 15.0-16.0years; his brother and maternal uncle also had delayed onset of puberty and growth spurt.Both the proband and his brother had spontaneous pubertal development without testosterone therapy on follow up, thus excluding HH.The second male proband (B) presented at 14.5-15.0years with delayed pubertal onset (testicular volumes 3.5 mL and lack of secondary sexual characteristics).He had a family history of delayed puberty in his mother and maternal grandfather.Proband C, also male, was first seen at age 15.0-15.5 years with testicular volumes of 4.5 mL and bone age delay of 2.8 years.Both probands B and C had progressed through spontaneous puberty by the age of 18 years, confirming a self-limited DP phenotype.DNA was not available from family members of these two probands.
The SEMA6A I423T variant is predicted to affect protein stability The identified SEMA6A variant was not found in the gnomAD browser (accessed 28.06.2023).The substitution of the non-polar amino acid isoleucine (Ile, I) with a neutral polar amino acid, threonine (Thr, T), is predicted to be damaging to protein function or disease-causing by 3/3 bioinformatic tools (Poly-Phen, SIFT and REVEL) with a combined annotation dependent depletion (CADD) score of 31 (Fig. 8b).The affected I423 residue lies in the SEMA domain and is highly conserved between species (Fig. 8c, d).
decrease in protein stability.Therefore, the impact of Ile to Thr substitution on protein stability of both SEMA6A chains was evaluated in silico with the BioLuminate Residue Scan tool.This analysis showed that the stability of the mutated SEMA6A homodimer was substantially decreased compared to the WT SEMA6A homodimer (ΔStability: 16.22 ± 0.14 kcal/mol for each single SEMA6A protomer), supporting an effect of the identified variant on SEMA6A stability.

The SEMA6A I423T variant affects protein levels and localization in vitro
To functionally validate the in silico modeling predictions, we generated myc-tagged vectors encoding WT and mutated forms of human SEMA6A (SEMA6A WT and SEMA6A I423T respectively) followed by expression in COS-7 cells.Then, protein synthesis and cellular localization were analyzed as previously described for other semaphorins at 48 h post-transfection 12,18 .Western blotting analysis revealed that both SEMA6A WT and SEMA6A I423T proteins were successfully synthetized, although expression of the mutant protein was significantly reduced as compared to SEMA6A WT (Fig. 8h, i; p < 0.0001; two-tailed unpaired Student's t test).This suggests that the mutation affects protein expression and/or stability.
To examine whether the variant could also affect protein localization, we performed immunofluorescence using an anti-c-myc antibody to visualize SEMA6A WT or SEMA6A I423T in transfected cells (Fig. 8j).SEMA6A WT protein was detected at the cell membrane, as previously reported 61 , while SEMA6A I423T protein mainly localized in the perinuclear region.To understand the subcellular localization of mutant SEMA6A, COS-7 cells were co-transfected with SEMA6A I423T and a fluorescent endoplasmic reticulum (ER)-marker (mEmerald-ER-3).
High level of signal colocalization suggests that SEMA6A I423T was retained in the ER (Fig. 8k).Together, the in vitro results strongly support our in silico predictions and indicate that the I423T variant might affect SEMA6A stability and impair intracellular protein trafficking.The consequently defective membrane localization would hamper any paracellular interaction with SEMA6A receptors, suggesting a loss-of-function effect of the SEMA6A I423T variant in patients.

Discussion
Our study provides insights into the mechanisms that regulate puberty onset by implicating SEMA6A in the control of ME homeostasis and GnRH neuron plasticity and by identifying human patients with delayed onset of puberty carrying a pathogenic SEMA6A variant.Similar to other members of the semaphorin family, SEMA6A regulates axon guidance during brain development 25,62 .In line with this, we detected strong SEMA6A expression in nasal axons of developing mouse and human embryos (Fig. 1).However, SEMA6A loss did not affect GnRH axon pathfinding or GnRH neuron migration (Fig. 2).These results indicate a dispensable role for SEMA6A in these processes, as already observed for other semaphorins (e.g., SEMA3F) 11 .In addition to their key roles in axon guidance and neuronal migration at embryonic stages, semaphorins are expressed in the postnatal hypothalamus where they control GnRH neuron structural plasticity [15][16][17] .We found that SEMA6A is stably expressed in the ME parenchyma of perinatal (E18.5) and postnatal mice (P24 and P60) (Figs. 1, 3), a region that undergoes several cellular rearrangements to regulate neurohormonal homeostasis 39 .Intriguingly, neither SEMA6A transcript nor protein were detected in neuroendocrine neurons, β-tanycytes or astrocytes (Fig. 5 and Supplementary Fig. 4).These cells are known to play key roles in GnRH neuron plasticity 43,44 .Instead, we identified maturing OLs as the source of SEMA6A in the ME (Fig. 5 and Supplementary Fig. 4).Thus, in addition to its previously described role in OL differentiation 45 , SEMA6A derived from OLs in the ME serves as a signal for regulating ME vascular permeability and GnRH neuron plasticity.
Sema6a -/-mice displayed decreased GnRH axon innervation of the ME starting from E18.5 (Fig. 2), when the first GnRH + fibers project to the ME 35 , until adult stages (P60) (Fig. 4), when GnRH neuron plasticity must be tightly regulated to guarantee proper GnRH secretion 1 .In line with these observations, both male and female Sema6a -/-mice showed delayed puberty onset and sexual maturation defects (Fig. 4).The decrease in GnRH neuron innervation of the ME was not accompanied by a reduction in the number of GnRH neurons reaching the MPOA (Fig. 2).This excludes a decrease in neuronal number as the cause for the reduced density of GnRH-positive axons in the ME.Moreover, our data also argue against a direct effect of SEMA6A on GnRH neuron axons, as no expression of the SEMA6A receptors Plexin-A2 and Plexin-A4 was detected in MPOA-resident GnRH neurons (Supplementary Fig. 2).
The expression of SEMA6A in the external portion of the ME adjacent to fenestrated blood capillary loops invading the brain parenchyma, suggested a possible role in the NGE unit.This is a fundamental structure formed by a dynamic network of capillaries, βtanycytes and neuroendocrine projections 5,39 .Interestingly, previous studies have described roles for semaphorins, such as SEMA3A and SEMA7A, in the control of GnRH neuron plasticity by modulating GnRH neurite extension 15 or tanycyte endfeet sprouting 17 , respectively.However, evidence for direct control of ME vascular permeability by semaphorins had not been reported.Here, by combining in vitro vascular permeability experiments and in vivo functional studies, we unveiled a thus far undiscovered role for SEMA6A in ME vascular barrier homeostasis.Specifically, we found that a secreted form of SEMA6A increases permeability of cultured EC monolayers (Fig. 6 and Supplementary Fig. 5), and that Sema6a -/-mice display a less permissive ME vascular barrier.This was associated with fewer fenestrated loop capillaries, but normal β-tanycyte morphology (Fig. 7 and Supplementary Fig. 6).These findings strongly support a direct role for SEMA6A in the regulation of ME vascular permeability.
The effect of SEMA6A on ECs is likely to be mediated by its canonical receptor Plexin-A2.Plexin-A2 is expressed by fenestrated capillaries of the ME in vivo (Fig. 7) and PLXNA2 knockdown attenuated SEMA6A-induced vascular permeability in vitro (Fig. 6).These data show that SEMA6A can act via Plexin-A2 to modulate vascular permeability.Still, we cannot exclude that SEMA6A also interferes with Vascular endothelial growth factor receptor 2 (VEGFR-2) signaling, as previously reported in HUVECs 29,63 , possibly through the binding with other SEMA6A interactors such as members of the Ena/VASP protein family 33,64 .VEGFR-2 is expressed by fenestrated ECs and has previously been shown to sustain ME vascular permeability through VEGF-A signaling 57 .
Our findings provide support for a key role of SEMA6A in the regulation of NGE unit.The NGE unit regulates the outflux of neurohormones, including GnRH, to target tissues, as well as the influx of molecules from peripheral tissues.Therefore the ability of SEMA6A to regulate ME vascular barrier could modulate trafficking of molecules leading to changes in factor secretion or in uptake of circulating peptides 39,65 .In particular, decreased ME vascular permeability in the absence of SEMA6A (Fig. 7) may reduce the uptake of circulating factors that regulate GnRH neurite extension and/or GnRH secretion.For example, blood-borne Insulin-like growth factor 1 (IGF-1) and Fibroblast growth factor 21 (FGF-21) can induce the release of GnRH 66 and stimulate GnRH neurite outgrowth with subsequent peptide release 40 , respectively (Fig. 9).Our observation that loss of SEMA6A reduces the diffusion of EB dye from the periphery through the ME barrier supports this model.Of note, mice lacking IGF-1 or FGF-21 receptors also exhibit DP phenotypes like Sema6a -/-mice 40,67 .
The identification of a predicted pathogenic variant in SEMA6A in 6 individuals with a DP phenotype serves to translate our experimental observations to the clinic and provides a mechanistic link between SEMA6A deficiency and GnRH functionality in humans.DP is a highly heritable condition affecting up to 2% of the population 8,68 .Yet, despite recent progress, the genetic determinants of DP remain largely unknown 10 .The analysis of exome sequencing from 100 probands from a self-limited DP cohort led to the identification of this novel missense variant in SEMA6A, segregating with the DP trait in 4 individuals from the same family and two further probands from unrelated families.Interestingly, our patients do not exhibit other pituitary hormone deficiencies, strongly supporting a selective effect of SEMA6A on the regulation of the HPG axis.Our work provides strong support for a deleterious effect of this SEMA6A I423T variant on protein stability and localization.The DP patients in our cohort are heterozygous carriers of the SEMA6A I423T variant, whilst in the mouse model Sema6a -/-but not Sema6a +/-mice displayed DP.However, previous work on several related genes has demonstrated that even when patients carry heterozygous pathogenic mutants, only full knock-out, but not heterozygous, mice targeting the corresponding genes exhibit a pathological reproductive phenotype (e.g., Sema3a, Sema7a, Plxna1) 11,14,69 .The same phenomenon has been demonstrated for other developmental genes 70 .This would suggest that patientderived mutations can exhibit a dominant effect on protein function, such as the altered stability and localization of the SEMA6A I423T In conclusion, our results uncover an additional role for the axon guidance cue SEMA6A in the control of puberty onset and sexual maturation and help to understand the pathogenic mechanisms underlying a novel SEMA6A variant in patients affected by DP.Specifically, these data unveil an additional mechanism through which OLproduced SEMA6A acts on Plexin-A2 + ECs to regulate ME homeostasis and, as a consequence, GnRH neuron innervation.Our findings provide a framework for further understanding the genetic and molecular mechanisms that underlie puberty onset as well as for guiding diagnostic and therapeutic approaches in pubertal delay.Further, the outlined mechanism of disrupted ME vascular permeability could potentially have broader implications on various neuroendocrine functions beyond pubertal maturation.

Ethics
The human fetal tissue was provided by the Joint MRC/Wellcome Trust (grant #099175/Z/12/Z) Human Developmental Biology Resource (www.hdbr.org),with appropriate maternal written informed consent and approval from the Ethics Committee NRES Committee London -Fulham.HDBR is regulated by the UK Human Tissue Authority (HTA; www.hta.gov.uk) and operates in accordance with the relevant HTA Codes of Practice.Fetal tissues from both sources were obtained from terminations at 7 post conception weeks (Carnegie Stage 19).The human study protocol was approved by the Ethics Committee for Pediatrics, Adolescent Medicine and Psychiatry, Hospital District of Helsinki and Uusimaa (570/E7/2003).Ethical approval for human studies was granted by the UK London-Chelsea NRES committee (13/LO/0257).All participants provided written informed consent prior to study participation.The study was conducted in accordance with the guidelines of The Declaration of Helsinki.Participants did not receive compensation.

Animals and tissue preparation
HA-Sema6a fl/fl mice, which harbor a 3xHA tag in exon 2 of Sema6a and lox sites surrounding exon 3, were a kind gift of Alex Kolodkin (Johns Hopkins University School of Medicine, Baltimore, USA).Germline deletion of Sema6a exon 3 was performed by crossing these mice with EIIa-Cre mice (JAX stock #003724) 71 to create Sema6a-null mice (here referred to as Sema6a -/-) that were maintained on a C57Bl/6J background (JAX strain, strain code 632, Charles River).Genotyping was performed by PCR using DNA from mouse tail or ear tissue as a template and primers flanking the HA tag sequence (fw '5-Fig.9 | SEMA6A modulates GnRH neuron homeostasis by tuning ME vascular permeability.Schematic drawing representing the proposed cellular mechanisms through which SEMA6A is expressed by OLs to modulate GnRH homeostasis.In the ME of WT mice, SEMA6A (green dots) is produced by OLs (green cells) and distributed close to fenestrated ECs (red cells).In our model, SEMA6A acts on fenestrated ECs expressing Plexin-A2 (blue receptors) to increase the number of capillary loops in the brain parenchyma and maintain a permissive vascular barrier (1).Such permissive vascular barrier may ensure optimal GnRH peptide (gray dots) release in ME blood vessels either directly (2) or indirectly, via entry of molecules from the periphery (e.g., IGF-1, FGF-21; black triangles) in the ME (3)   where they can act on GnRH neurons (in gray), by modulating the extension of axon terminals towards ME and ECs (4).This mechanism will ensure a normal release of GnRH peptide (5) (gray dots) and therefore a physiological puberty onset.Conversely, in Sema6a -/-mice, the lack of SEMA6A production by OLs (gray cells) in the basal part of the ME leads to fewer capillary loops and reduced vascular permeability.Consequently, direct GnRH release to the pituitary or entry of peripheral signals that normally sustain axon terminal elongation and GnRH secretion do not occur, resulting in a less innervated ME and ultimately in delayed puberty onset.Created with BioRender.com.
CCTTGTCACACATGCAGTTG-3' and rev '5-TGAAGTAGAGAAGCA-3'), and by primers controlling for germline deletion of exon 3 (fw '5-GCTCTTTGGCAGGGTGAAGTA-3' and rev '5-GTCCAAGTCCACGT-GACCC-3').For all experiments, mice were housed socially with chow diet (CRM (E) SQC 811437, Special Diet Services) and water ad libitum, on a 12 h light/dark cycle, humidity of 45-65% and a temperature between 20 and 24 °C.The morning of vaginal plug observation was defined as embryonic day 0.5 (E0.5).The day of birth was defined as postnatal day 0 (P0).Timed pregnant females were sacrificed by cervical dislocation to collect embryos at E12.5, E14.5 and E18.5.Embryo heads were fixed for 3-6 h in 4% PFA at 4 °C.Adult mice were euthanized by overdosing Euthanimal (200 mg/mL, Alfasan) intra-peritoneal (i.p.), and transcardially perfused with ice-cold 4% PFA.Subsequently, brains were isolated and post-fixed in 4% PFA overnight at 4 °C.All samples for cryosectioning were cryoprotected overnight in 30% sucrose prior to OCT-embedding, whereas samples for paraffin embedding were dehydrated by immersion through crescent alcohol series and xylene.All in vivo experiments were conducted by certified personnel in compliance with the Animal Ethics Committee of Utrecht University (Dierexperimenten Ethische Commissie) (CCD licence: AVD115002016532 and AVD11500202114777) and the Animal Welfare Body of the University of Milan, and in agreement with Dutch laws (Wet op de Dierproeven, 1996; revised 2014), the Italian Minister of Health and European regulations (Guideline 86/609/EEC; Directive 2010/63/ EU).Animal welfare was checked daily.
Hematoxylin & eosin staining and follicle quantification 8 µm microtome sections of PFA-fixed paraffin-embedded ovaries were rehydrated with xylene/decrescent alcohol series, stained with Mayer's hematoxylin (Bio-Optica) for 2 min and Eosin Y alcoholic solution (Bio-Optica) for 3 min and finally dehydrated with crescent alcohol series/ xylene.Two sections for each female mouse were considered for the count of ovarian follicles.Follicles were distinguished into primary (single layer surrounding the oocyte), secondary (two layers surrounding the oocyte), pre-antral (three or more layers surrounding the oocyte) and early antral (three or more layers surrounding the oocyte with the presence of a cavity) and quantified 83 .
Determination of male balanopreputial separation, female vaginal opening and first estrous Sema6a -/-, Sema6a +/-, and Sema6a +/+ male and female mice were weighed and checked daily for BPS and VO 37,38,84 , respectively, after weaning from P22 onwards.When female mice showed VO, vaginal smears were obtained every 24 h to assess the start of the estrous cycle by gently pipetting and resuspending 30 µL saline solution (0.9% NaCl) in the VO using a P200 pipette.The collected cells were transferred to a glass microscope slide and air-dried.Slides were then treated with 70% EtOH for 3 min, followed by 0.1% Cresyl Violet stain in 50% EtOH for 1 min, and briefly washed with 100% EtOH.Subsequently, smears were evaluated using light microscopy and the relative amount of nucleated, anucleated epithelial cells and leukocytes present was used to determine the phase of the estrous cycle (Supplementary Fig. 3c) 37 .Gross absence of leukocytes and presence of multitudes of anucleated epithelial cells was considered as estrous.Tissues for analysis were collected from male and female mice at the day of BPS and FE, respectively.Mice were euthanized by cervical dislocation, brains and gonads were isolated and fixed overnight in 4% PFA at 4 °C, and pituitaries were snapfrozen and stored at −80 °C.Males were collected from 13 litters, females were collected from 14 litters.

Evans Blue (EB) in vivo permeability assay
At P30, Sema6a -/-, Sema6a +/-, and Sema6a +/+ female mice were euthanized by overdose of Euthanimal i.p. (200 mg/mL, Alfasan).Subsequently, animals were transcardially perfused with 20 mL sterilefiltered 68 mg/mL EB dye (Sigma-Aldrich) dissolved in 4% BSA in PBS, followed by perfusion with 25 mL PBS.Perfusion speed was kept at 3 mL/min.Following perfusion, brains were isolated and cryoprotected by incubation in ascending concentrations of sucrose (10%-20%-30%) in PBS for 30 min each at 4 °C.Brains were then embedded in OCT and snap-frozen in dry-ice.Sema6a +/-and Sema6a +/+ females who did not yet show VO at P30 were excluded from analysis.WT and Sema6a mutant animals were collected from 4 litters.For analysis, 30 µm cryosections were directly imaged under laser scanning confocal microscope to visualize EB staining excited at 640 nm.EB diffusion in the ME parenchyma was assessed by quantifying the background-normalized mean gray values of three equally sized fields per section, randomly placed in the ME region, using ImageJ software (v1.52a,NIH).

scRNA-seq analysis
Raw data for adult brain scRNA-seq datasets from Tabula Muris and EC atlas were obtained from NCBI gene Expression Omnibus (GSE109774) and EBI ArrayExpress (E-MTAB-8077), respectively, and analyzed with RStudio v.1.3.1056.The raw gene expression matrices (UMI counts per gene per cell) were filtered, normalized and clustered using the R package Seurat v.3.2.0.Cells containing less than 200 feature counts and genes detected in less than 3 cells were removed.Downstream analysis included data normalization ("LogNormalize" method and scale factor of 10,000) and variable gene detection ("vst" selection method, returning 2000 features per dataset).The principal components analysis (PCA) was performed on variable genes, and the optimal number of principal components, PCs, for each sample was chosen using the elbow plot.The selected PCs were used for Louvain graph-based clustering at a resolution of 0.3 followed by manual curation based on marker genes to obtain the final seven and four clusters from Tabula Muris and EC atlas brain datasets, respectively.Uniform manifold approximation and projection (UMAP) was chosen as a non-linear dimensionality reduction method, and each relevant gene was then examined using the FeaturePlot and VlnPlot functions.Cluster cell identity was assigned by manual annotation based on known marker genes.Clustering was then manually curated based on marker genes to obtain the final seven and four clusters from Tabula Muris and EC atlas brain datasets, respectively, followed by subset selection in the Tabula Muris dataset of the cluster containing Emcn + ECs.
Cell culture, transfection and lentivirus infection COS-7 cells (American Type Culture Collection, cat.CRL-1651) were grown as a monolayer at 37 °C in a humidified CO 2 incubator in complete DMEM (Euroclone) and supplemented with 10% fetal bovine serum (FBS; Gibco).Subconfluent cells were harvested by trypsinization and cultured in 57 cm 2 dishes.For transfection, COS-7 cells (at 80% confluence) were grown in culture plates in complete culture medium for 24 h and incubated for 48 h with the selected expression vector (1 μg/mL) in the presence of Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions.To visualize Endoplasmic Reticulum (ER), the mEmerald-ER-3 vector (gift from Michael Davidson; Addgene plasmid #54082) was cotransfected with the SEMA6A I423T vector (ratio 1:4).The conditioned media of COS-7 cells either mock transfected or expressing SEMA6A ECTO were collected after 48 h and stored at −80 °C.HUVECs from pooled donors (Promocell, cat.FB60C12203) were cultured on fibronectin (Promocell) or rat collagen I coated vessels (Corning, cat.DLW354236) in EGM-2 complete medium (Promocell).To generate stable PLXNA2 knockdown cells, HUVECs were separately infected with a set of lentiviral particles expressing four different shRNAs (A-D) against human PLXNA2 (Origene, cat.TL302405V) with a calculated MOI of 10; cells infected with lentiviral particles expressing scramble shRNA were used as controls.At 48 h post infection, cells were selected with 1 μg/mL puromycin.Knockdown efficiency was evaluated by RT-qPCR.

Plasmids
The pAPtag-5 vector (6.6 kb) (GenHunter, cat.Q202) was chosen for the cloning of the shorter isoform of human SEMA6A gene (NM_020796.5),encoding for a protein of 1030 amino acids (expected molecular weight 120 kDa).Two different vectors have been prepared with human SEMA6A WT and SEMA6A I423T genes inserted between the NheI and the XhoI restriction enzyme sites, in frame with c-myc tag, cutting off the AP protein.The empty vector has been used as a control vector.The pCAGG-AP-SEMA6A-ecto-Fc-His vector was employed to produce mouse SEMA6A soluble extracellular domain ranging from amino acid 18 to 688 (SEMA6A ECTO ) 31 .
Isolation and culture of ECs from adult mouse brain mBECs were isolated from 3 to 5 brains of adult WT C57Bl/6 mice by slightly modifying an existing protocol 52 .Briefly, brains were cut along the longitudinal fissure to separate the two hemispheres and the cerebellum was removed from the rest of the brain.The meninges were removed by rolling the individual parts on 3 M paper.Brain hemispheres were cut and minced using a scalpel, resuspended in dissection buffer (Optimem, 2% FCS, 2 ng/mL heparin and pen/strep), and passed through a 10 mL serological pipet until no evident clumps were present.The homogenate was centrifuged (300 g, 5 min) and the pellet was digested in dissection buffer containing 1 mg/mL of collagenase/ dispase (Sigma Aldrich) and 50 µg/mL of DNase (Sigma Aldrich) on a benchtop shaker (900 RPM) for 45 min at 37 °C.To remove the myelin, the pellet was resuspended in 22% (w/v) of bovine serum albumin (BSA) in PBS and centrifuged (1620 g for 15 min at 4 °C) for 3 times.The combined pellets were then resuspended in EGM complete medium (Promocell) supplemented with 5 µg/mL of puromycin to kill non-ECs and seeded on collagen coated (rat tail collagen I 50 µg/mL in PBS; Corning, cat DLW354236) 0.4 μm pore-size Transwell Permeable Supports (Merck, cat.MHCT24H48).Culture medium was replaced after 48 h with complete medium and changed every other day until confluence.mBECs purity was evaluated by morphological inspection and immunofluorescence staining for the EC markers PECAM1 and Cadherin-5 as described below.

ECs permeability assay
HUVECs or mBECs were seeded on fibronectin or collagen I coated 0.4 μm pore-size Transwell Permeable Supports (Merck, cat.MHCT24H48), cultured in complete culture EGM-2 or EGM medium (Promocell), respectively, and let to form a monolayer.After the establishment of a stable monolayer, cells were starved overnight with DMEM without phenol red supplemented with 2% FBS and antibiotics.The next day, HUVECs or mBECs were stimulated with conditioned medium containing murine SEMA6A ECTO or a mock control added in the apical or basal side of the transwell.DMEM without phenol red supplemented with 2% FBS with and without VEGF-A (50 ng/mL) was used as negative and positive control, respectively.TEER was assessed using a Millicall-ERS Voltohmeter (World Precision Instruments) 87,88 .TEER measurements were performed using the same conditions to allow comparison of different groups and calculated as area-integrated resistance (Ωcm 2 ).The percentage of TEER change was calculated with respect to time 0 for each indicated time point.

Human genetic analysis
Patients from a large Finnish cohort with self-limited DP were analyzed for this study.Patients referred with self-limited DP to specialist pediatric care in central and southern Finland between 1982 and 2004 were identified.All participants (n = 492) met the diagnostic criteria for self-limited DP 89 .Inclusion criteria were the onset of Tanner genital stage II (testicular volume > 3 mL) > 13.5 years in boys or Tanner breast stage II > 13.0 years in girls (i.e., two SD later than average pubertal development) and their unaffected relatives.Normal values of thyroid function tests (TFTs) and IGF-1 for bone age were also used as inclusion criteria.Chronic illness as a cause for functional HH was excluded by medical history, clinical examination, and biochemical investigations.Congenital or acquired HH, if suspected, was excluded by spontaneous pubertal development by 18 years of age at follow-up.Sex was considered in the study design, and sex of participants was determined based on self-report.

Whole exome sequencing analysis
Whole exome sequencing was performed on DNA extracted from peripheral blood leukocytes of 100 probands, using an Agilent V5 platform and Illumina HiSeq 2000 sequencing.The exome sequences were aligned to the UCSC hg19 reference genome using the Burrows-Wheeler Aligner software (BWA-MEM [bwa-0.7.12]).Picard tools (picard-tools-1.119)was used to sort alignments and mark PCR duplicates.The genome analysis toolkit (GATK-3.4-46) was used to realign around indels and recalibrate quality scores using dbSNP, Mills and 1000 genomes as reference resources.Variant calling and joint genotyping using pedigree information was performed using Haplotype-Caller in GVCF mode from the genome analysis toolkit.The resulting variants were filtered using the variant quality score recalibration (VQSR) function from GATK.An analysis of the called variants was performed using Ingenuity Variant Analysis (QIAGEN Redwood City, www.qiagen.com/ingenuity).Filtering for potential causal variants was carried out using filters for quality control (read depth and Phred strand bias), minor allele frequency (MAF < 0.5% in the ExAC and Genome Aggregation Database (gnomAD) databases v2.0.2), predicted functional annotation (Poly-Phen, SIFT, REVEL, CADD score) and conservation score (GERP).Potential causal variants were confirmed by Sanger sequencing.

In silico mutagenesis
The CryoEM structure of the SEMA6A homodimer in complex with C. sordellii lethal toxin TcsL (PDB ID: 6WTS; https://doi.org/10.2210/pdb6WTS/pdb) 59 was retrieved from the RCSB Protein Data Bank.After a structure preparation procedure via Schrödinger BioLuminate Protein Preparation Tool, including an energy minimization using the OPLS3e force field 90 , the TcsL toxin was removed.The evaluation of the impact of I423T mutation on the protein stability has been carried out with the Schrödinger BioLuminate Residue Scanning Tool.Stability is computed from a thermodynamic cycle which considers the relative stability of each entity: unfolded SEMA6A, folded SEMA6A, unfolded mutated SEMA6A and folded mutated SEMA6A 91 .
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Fig. 1 |
Fig. 1 | SEMA6A is expressed in territories relevant for embryonic GnRH neuron development.Coronal sections of E12.5 (a) and E14.5 (b) mouse heads at the nose level were immunolabelled for SEMA6A (green) and Peripherin (red).Solid arrowheads indicate examples of Peripherin-positive axons that express SEMA6A at the VNO level and in the nasal parenchyma.Coronal sections of E12.5 (c) and E14.5 (d) mouse heads at the nose level were immunolabelled for SEMA6A (green) and GnRH (red).Empty arrowheads indicate examples of GnRH neurons that lack SEMA6A expression at the VNO level and in the nasal parenchyma.Coronal sections of E12.5 (e) and E14.5 (f) mouse heads at the nose level were immunolabelled for SEMA6A (green) and BLBP (red).Empty arrowheads indicate examples of OECs that lack SEMA6A expression at the VNO level and in the nasal parenchyma.Coronal sections of E18.5 mouse brains at the MPOA (g) and ME (h) levels were immunolabelled for SEMA6A (green) and GnRH (red).Empty arrowheads indicate examples of GnRH neurons that lack SEMA6A expression in the cell body (g) and neurite (h).Sagittal view of a CS19 human embryo in a schematic drawing (i) and sagittal sections of schematics squared area immunolabeled for SEMA6A (j-m).Black arrows indicate examples of SEMA6A-expressing axons in the nasal parenchyma.Sections in (a-h) were counterstained with DAPI.Dotted boxes indicate areas shown at higher magnification next to the corresponding panel.Abbreviations: VNO vomeronasal organ, NS nasal septum, FB forebrain, OE olfactory epithelium, OB olfactory bulb, MPOA medial preoptic area, ME median eminence, 3v third ventricle, CS Carnegie Stage.Scale bars: 500 μm (j), 250 μm (k), 200 μm (a, c, e, g, low magnifications), 125 μm (l, m), 100 μm (h, low magnification), 50 μm (a, c, e, g, h, high magnifications).

Fig. 2 |
Fig. 2 | SEMA6A loss does not impair nasal axon patterning and GnRH neuron migration, but reduces GnRH innervation of the ME. a Coronal sections of E14.5 mouse heads of the indicated genotypes at the level of the nose and forebrain were immunolabeled for Peripherin (green) to mark nasal axons.White arrows indicate normal nasal axon development in the nose and normal TN fibers in the MPOA.Sections were counterstained with DAPI.b Coronal sections of E14.5 mouse heads with the indicated genotypes were immunolabelled for GnRH.Black arrows indicate examples of normal GnRH neuron distribution in the nasal parenchyma at the CP and in the forebrain.c Quantification of the distribution and total number of GnRH neuron in E14.5 heads (n = 4 per group; nose p = 0.7974, CP p = 0.6360, forebrain p = 0.8596, TOT p = 0.5670).d Quantification of GnRH neuron number in E18.5 brains (from OB to MPOA) (Sema6a +/+ : n = 3; Sema6a -/-: n = 5; p = 0.7819).e Coronal sections of E18.5 brains with the indicated genotypes at the level of the ME were immunolabelled for GnRH.Black arrows indicate normal presence of GnRH neuron axon terminals in Sema6a +/+ embryos, whereas the decreased presence of GnRH neuron axon terminals is indicated with Δ. f Quantification of GnRH intensity at ME in E18.5 brains (Sema6a +/+ : n = 3; Sema6a -/-: n = 5; p = 0.0189).*p < 0.05 and NS (not significant) after two-tailed unpaired Student's t test.Abbreviations: VNO vomeronasal organ, OE olfactory epithelium, OB olfactory bulb, CP cribriform plate, FB forebrain, MPOA medial preoptic area, 3 v third ventricle.Scale bars: 200 μm (a), 125 μm (b, e).Data are presented as mean ± SD.Source data are provided as a Source Data file.

Fig. 3 |
Fig. 3 | SEMA6A is expressed in the postnatal ME in close proximity to GnRH neuron terminals.Coronal sections of P24 (a) and P60 (b) ME were immunolabelled for SEMA6A (green) and GnRH (red) to reveal GnRH axon terminals.White boxes in (a, b) indicate areas shown at higher magnification in the adjacent panels together with single channels for SEMA6A and GnRH.Empty arrowheads indicate examples of GnRH neuron axon terminals that lack SEMA6A expression.White arrows indicate the expression of SEMA6A in the basal part of the ME.All sections were counterstained with DAPI.Abbreviations: 3v, third ventricle.Scale bars: 100 μm (low magnifications), 50 μm (high magnifications).

Fig. 5 |
Fig. 5 | SEMA6A is detected in OLs in the ME and not by other cell types composing the NGE unit.a-e Coronal sections of adult ME were immunolabelled for SEMA6A (green) together with IB4, Synaptophysin, GFAP, Vimentin and Oligo2 (red) to label blood vessels, neuroendocrine neurons, astrocytes, tanycytes and oligodendrocytes, respectively.White boxes indicate areas shown at higher magnification next to each corresponding panel together with single channels for SEMA6A and different cell markers.Empty arrowheads indicate examples of blood vessels penetrating in the ME that lack SEMA6A expression (a) and the lack of SEMA6A expression on neuroendocrine axon terminals (b), astrocytes (c), tanycytes (d).Solid arrowheads indicate the expression of SEMA6A on oligodendrocytes (e).All sections were counterstained with DAPI.Abbreviations: 3v third ventricle.Scale bars: 100 μm (a, b low magnifications), 50 μm (a, b, high magnifications; c-e, low magnifications), 25 μm (c, d, high magnifications), 12 μm (e, high magnification).

Fig. 7 |
Fig. 7 | SEMA6A loss prevents ME barrier opening and induces structural rearrangements in the ME capillary bed. a Coronal sections of adult ME were immunolabelled for Plexin-A2 (green) together with PECAM1 (red) to label blood vessels.The white box indicates the area shown at higher magnification next to each corresponding panel together with single channels for Plexin-A2 and PECAM1.Solid arrowheads indicate the expression of Plexin-A2 on capillaries of the basal portion of the ME.b Coronal sections of brains from EB-injected females of the indicated genotypes.White boxes indicate areas shown at higher magnification next to each corresponding panel.Δ indicates areas of no EB leakage in Sema6a -/- ME parenchyma.c Quantification of EB diffusion in the ME parenchyma of female brains (n = 5 per group; p = 0.0212).d Coronal sections of female brains with the indicated genotypes were immunolabelled for PLVAP (green) to reveal capillary loops invading the ME parenchyma.White boxes indicate areas shown at higher magnification next to each corresponding panel together with a single channel for PLVAP.White arrows or Δ indicate the normal or decreased presence of capillary loops in Sema6a +/+ and Sema6a -/-ME parenchyma, respectively.e Quantification of capillary loops in the ME of female brains (n = 3 per group; p = 0.0230).All sections were counterstained with DAPI.*p < 0.05 after two-tailed unpaired Student's t test.Abbreviations: 3v third ventricle, EB Evans Blue.Scale bars: 100 μm (b, d low magnifications), 50 μm (a, low magnification; b and d, high magnifications), 25 μm (a, high magnification).Data are presented as mean ± SD.Source data are provided as a Source Data file.

Fig. 8 |
Fig. 8 | Exome sequencing identifies a SEMA6A variant with effects on protein stability in DP patients.a Family tree of proband A (III.2, arrow) presenting DP.Symbols: DP phenotype (black), unknown phenotype (gray); unaffected individual (white).Horizontal black lines indicate the presence of heterozygous p.I423T variant identified by WES and verified by Sanger sequencing.US signifies that DNA was unavailable for sequencing.b Chromosome (Chr) position, nucleotide substitution (nt sub), amino acid substitution (aa sub), and bioinformatic predictions of the p.I423T SEMA6A variant according to Poly-Phen, SIFT, REVEL and CADD software.c Partial protein sequence alignment of vertebrate SEMA6A orthologs shows that I423 conservation in mouse and zebrafish.d Genomic evolutionary rate profiling of sequence constraint for SEMA6A variant using GERP + + analysis (RS score = 6.0).Side (e) and top (f) 3D views of modeled human SEMA6A monomer.Red asterisks indicate the I423 residue within the central cavity of SEMA domain 6th betapropeller blade.g High magnification images of I423 and T423 residues and their interactions.h Lysates from untransfected (UT) COS-7 cells and cells transfected with an empty control expression vector (CTRL) or vectors encoding c-myc tagged human SEMA6A WT or mutant SEMA6A I423T were immunoblotted for c-myc to recognize human (SEMA6A: 120 kDa).GAPDH (37 kDa) was used as a loading control.i Quantification of relative abundance of SEMA6A I423T protein compared to SEMA6A WT after normalization with GAPDH (n = 3 independent experiments; p < 0.0001).j COS-7 cells transfected with vectors encoding c-myc tagged SEMA6A WT or SEMA6A I423T were immunolabeled for c-myc (green) and F-actin (red).Solid arrowheads indicate cell surface SEMA6A WT expression, whereas empty arrowheads indicate lack of surface SEMA6A I423T but intracellular and perinuclear expression.k COS-7 cells co-transfected with vectors encoding SEMA6A I423T and ERemerald, visualizing ER (green), were immunolabeled for c-myc (red) and counterstained with DAPI.Solid arrowheads indicate examples of SEMA6A I423T ER retention.****p < 0.0001 after two-tailed unpaired Student's t test.Abbreviations: ER endoplasmic reticulum.Scale bars: 50 μm (j, k).Data are presented as mean ± SD.Source data are provided as a Source Data file.

Table 1 |
Clinical data of auxological measurements and biochemical investigations from mid-childhood to adult age for the proband from the index family (proband A) variant.In contrast, gene targeting strategies in mice do not usually affect the stability of the protein encoded by the WT allele in heterozygous animals.